Cancer is the second leading cause of death in the United States after cardiovascular disease. One in three Americans will develop cancer in his or her lifetime, and one of every four Americans will die of cancer. Despite profound experience with the traditional therapeutic approaches—resection, radiation and chemotherapy, most of the malignant human tumors still follow a fatal clinical course. A novel development in tumor therapy is so called targeted therapy. This approach relies on the treatment of tumor initiating or promoting molecular alterations which are characteristic for the individual patient's tumor. Knowledge of such alterations might be decisive for the selection of the preferable kind of life prolonging or even curative therapy.
Mutations in the V-RAF MURINE SARCOMA VIRAL ONCOGENE HOMOLOG Bl (BRAF) have been described in multiple tumor series including melanoma (Davies et al. 2002 Nature 417: 949-54), papillary thyroid carcinoma (Basolo et al. 2010 J Clin Endocrinol Metab 95(9):4197-4205), colon cancer (French et al. 2008 Clin Cancer Res 14: 3408-15), ovarian cancer (Davies et al. 2002 Nature 417: 949-54), and other entities. BRAF is part of the RAS/RAF/MEK/ERK-signaling pathway which is involved in cell proliferation. Activation of this pathway results in promotion of malignant transformation.
One of the molecular targets of the targeted therapy approach for cancer therapy is an exchange of amino acid valin by amino acid glutamic acid in codon 600 of the human BRAF gene (V600E). Approximately 8% of all human tumors carry this particular mutation. The V600E mutation in BRAF renders the protein constitutively active. It is especially frequent in melanoma and thyroid tumors. Preclinical analyses indicated selective activity of RAF inhibitors in tumors with BRAF V600E mutation without affecting ERK signaling in normal cells (Poulikakos 2010, Nature 464: 427-30). Therapies targeting this V600E mutation with specific inhibitors are currently in clinical testing (Chapman 2009, European Journal of Cancer Supplements 7: 5).
The gold standard for diagnostic analysis of a BRAF V600E mutation is currently DNA sequencing. However, this method has some limitations: The percentage of tumor cells in the tissue taken for DNA extraction needs to be rather high. Too much contaminant with non tumorous cells will result in a false negative sequencing result. The procedure of DNA analysis is time consuming because at first, suitable material for DNA extraction must be outlined by a pathologist, DNA extraction needs to be performed and then, DNA can be sequenced. The present invention allows identification of even small tumor islets with the BRAF mutations within large areas of normal tissue without the mutation. The analysis of BRAF V600E mutation with the present invention can be performed within a few hours using the equipment available in routine pathology laboratories.